Measurement of adenylate cyclase activity is carried out by quantitative determination of cAMP produced from ATP as a substrate. Methods for measurement of cAMP are grouped into two methods using as a substrate (1) labeled ATP and (2) non-labeled ATP.
In the method using labeled ATP as a substrate (1), using ATP labeled by a radioactive element, for example, [α-32P] ATP, as a substrate, and cAMP ([32P] cAMP) generated from radioactively labeled ATP is separated and determined. See Y. Salomon et al., Anal. Biochem., 58, 541 (1974; R. A. Johnson et al., In Method in Enzymology, 195, 3 (1991)). The method employs sequential affinity chromatography with ionic exchange resin and aluminum oxide columns for separation of [32P] cAMP from [α-32P] ATP.
Although this method is sensitive, it relies upon dangerously and costly radioactively labeled compounds.
On the other hands, the methods using non-labeled ATP are classified into (1) radioimmunoassay wherein radioactively labeled cAMP is subjected to antigen-antibody reaction competitively with anti-serum including cAMP generated from non-labeled ATP and then radioactivity of binding antibody is assayed to determine cAMP content, and (2) protein-binding assay wherein radioactivity of 3H-cAMP bound with cAMP-dependent protein kinds is measured using specific binding between cAMP-dependent protein kinase and cAMP. See A. G. Gilman et al., Proc. Natl. Acad. Sci. USA, 67, 305 (1970).
The method using the substrate non-labeled ATP can not compensate decomposition of cAMP by cyclic nucleotide phosphodiesterase and therefore the method is not appropriate for samples including strong phosphodiesterase activity.
Given the safety and environmental concerns, the use of radioactive materials should be avoided. A need exists for a highly sensitive non-radioactive assay to measure adenylate cyclase activity and cAMP as an index of adenylate cyclase activity.
However, it is very difficult to determine cAMP without using radioactive compounds because of the extremely low concentration of cAMP in most mammalian issues. In addition, since non-cyclic adenine nucleotides such as cAMP, ADP and ATP in a biological sample are present in several hundred to several hundred thousand times the concentration of cAMP and also those chemical structures are similar to the that of cAMP, they act as interfering substances in assay of cAMP. Particularly, ATP is present in one hundred million times the concentration of cAMP and therefore it is substantially impossible to exactly determine cAMP without complete removal of endogenous ATP.
On the other hand, cAMP is converted into AMP by action of phosphodiesterase. An assay for AMP without radioactive compounds has been disclosed. Lowry et al. have developed a sensitive assay based on the fluorescence of reduced pyridine nucleotide. See O. H. Lowry et al., A Flexible System of Enzymatic Analysis, Harcourt Brace Jovanovich, New York (1972); F. M. Matschinsky et al., J. Histochem. Cytochem., 16, 29 (1968). The assay depends on it that absorbency of reduced nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm is 0.617 per 0.1 mmol and an absolute concentration of NADPH is calculated on absorbency of a sample.
An assay for AMP is disclosed which depends upon the stimulatory effects of AMP on glycogen phosphorylase, the enzyme that converts glycogen into glucose-1-phosphate in the presence of inorganic phosphate (P1). See E. Helmrich et al., Biochemistry, 52, 647 (1964); ibid., 51, 131 (1964); M. Trus et al., Diabetes, 29, 1 (1980). According to the method, glycogen phosphorylase activity is determined by an amount of glucose-1-phosphate generated from glycogen and AMP can be assayed using the glycogen phosphorylase activity as an index. Lurie also have developed a sensitive assay for AMP. See K. Lurie et al., Am. J. Physiol., 253, H662 (1987).
A method to increase the analytical sensitivity and specificity for cAMP or adenylate cyclase have employed enzymic degradatiom of non-cyclic adenine nucleotides or their removal by chromatography. See N. D. Goldberg et al., Anal. Biochem. 28, 523 (1969); B. Mcl. Breckenridge, Proc. Natl. Acad. Sci. USA, 52, 1580 (1964).
In the conventional analysis, since interfering endogenous ADP or ATP could not be completely removed, it has been considered that measurement of cAMP should be impossible. Therefore, there has been substantially no method for highly sensitive measurement of cAMP content and adenylate cyclase activity based on the amount of cAMP without using radioactive substances.
From a completely different point of view, the inventor and others previously attempted to develop assays for adenylate cyclase activity and cAMP and as a results of an intensive study, they had found a method for highly sensitive measurement of cAMP content and adenylate cyclase activity based on the amount of cAMP with using only enzymatic and chemical reactions, which comprises removing selectively interfering substances, endogenous non-cyclic adenine nucleotides, such as ATP, ADP and the like using enzymes, converting AMP into ATP, converting ATP into glucose-6-phosphate through fructose-6-phosphate, converting NADPH, determining NADPH concentration and correlating with cAMP concentration (WO94/17198).
According to the method, cAMP at an amount of μg order in a biological sample can be strictly measured at a level of pmol or fmol. See A. Sugiyama et al., Anal. Biochem., 218, 20 (1994); A. Sugiyama et al., J. Clin. Lab., 8, 437 (1994); A. Sugiyama et al., Anal. Biochem., 225, 368 (1995); A. Sugiyama et al., Yamanashi Med. J., 10, 11 (1995).
The reaction schemes of the conventional method above are described below.





The method as stated above was very excellent as a method for quantitative analysis in a principle of methodology and theoretically correct. However the method takes long time for a cleaning reaction or a reaction mixture gets cloudy when an enzyme has been deactivated by heating after a cycle reaction.